Polymicrobial infections are inherently more complex than mono-species infections, and this complexity has been a significant barrier to both a fuller appreciation of pathogenic mechanisms, and to the development of effective measures to control or prevent the disease. Periodontal diseases typify polymicrobial diseases, and are among the most common infections of humans. Although certain organisms, such as Porphyromonas gingivalis, are considered key pathogens, it is the polymicrobial community that initiates and drives the disease. In vivo imaging studies have shown that oral microbial communities are highly structured; however, little is known regarding the mechanisms that control spatial orientation within communities or the role of spatial structure in pathogenicity. Our ongoing studies have identified and quantified key spatial parameters in synergistic communities of the periodontal pathogen Aggregatibacter actinomycetemcomitans with S. gordonii. In this proposal, we will characterize the molecular basis and biological relevance of spatial parameters controlling the association of P. gingivalis with its community partners. In the first Aim, we will focus on the interaction between P. gingivalis and S. gordonii, a molecularly well-characterized process resulting in elevated pathogenicity of the dual species communities. We will determine the spatial parameters that define the dual species communities and their impact on pathogenicity. Further, through the use of specific mutants, we will assess the impact of known community virulence factors and communication mechanisms on spatial orientation. In the second Aim, we will use Tn-seq and RNA-seq genomic approaches to holistically assess the fitness determinants and transcriptome patterns which control spatial patterning in P. gingivalis and S. gordonii. We will prioritize potential contributors to metabolic synergy for further study of the relationship between spatial proximity and interspecies communication. In the third aim, we will explore Pg genetic pathways important for fitness during infection with a diverse set of co-infecting bacteria. The application of these focused and comprehensively based approaches will allow us to integrate key spatial parameters with molecular interspecies interactions in the context of community pathogenic potential. Successful completion of this project will provide fundamental information regarding the development and regulation of synergistic pathogenicity displayed by spatially defined polymicrobial communities which could ultimately be translated into therapeutic strategies designed to target the community-based pathogenesis that underlies periodontal disease.